Process for the preparation of 3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one and salts and solvates thereof

ABSTRACT

The invention relates to a novel process for the preparation of 3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one, a broad spectrum 5-HT receptor binding ligand having potent 5-HT 1A -agonistic as well as 5-HT 1D -antagonistic activity. The invention also relates to novel salts and solvates, in particular hydrates of salts of said compound, as well as to their use as medicaments. 
     The invention relates the compounds with the formula (7)

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/555,958, filed Mar. 25, 2004, the content of which is incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to a novel process for the preparation of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one, a broad spectrum 5-HTreceptor binding ligand having potent 5-HT_(1A)-agonistic as well as5-HT_(1D)-antagonistic activity. The invention also relates to novelsalts and solvates, in particular hydrates of salts of said compound.

The invention also relates to the use of a compound disclosed herein forthe manufacture of a medicament giving a beneficial effect. A beneficialeffect is disclosed herein or apparent to a person skilled in the artfrom the specification and general knowledge in the art. The inventionalso relates to the use of a compound of the invention for themanufacture of a medicament for treating or preventing a disease orcondition. More particularly, the invention relates to a new use for thetreatment of a disease or condition disclosed herein or apparent to aperson skilled in the art from the specification and general knowledgein the art. In embodiments of the invention specific compounds disclosedherein are used for the manufacture of a medicament useful in thetreatment of disorders in which serotonin receptors are involved, orthat can be treated via manipulation of those receptors.

BACKGROUND OF THE INVENTION

The protagonist of the present invention,3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one, is known as itshydrochloric acid salt. As such it is described as ‘example 8’ in EP0650 964. Despite the fact that the synthetic route as outlined in saidpatent has quite an acceptable yield, it is definitely not suited forsynthesis on the scale required for a drug in clinical development, letalone the scale required for a marketed drug. The problems with theoriginal synthesis are manifold. First, the key starting material,3-nitro-2-hydroxybenzaldehyde is not available on a commercial scale,and was prepared by nitration of 2-hydroxybenzaldehyde, resulting in amixture of 3- and 5-nitro-2-hydroxybenzaldehyde which can only beseparated by relatively complicated chromatographic methods. The secondstep, an Erlenmeyer condensation, results in a very viscous mixturewhich is hard to stir on a technical scale, and from which the isolationof the desired product appeared difficult as well. The third step, thereduction of the nitro group to the amine, using Fe/HCl is laborious,and the isolation of the reaction product from the large amount of rust(Fe₂O₃) appeared to be next to impossible on a technical scale. In step4 the potential carcinogen N-benzyl-bis-chloroethylamine was used. Largescale use of such compounds creates insurmountable safety hazards. Afteracidic amide hydrolysis (step 5) the end product is contained in a largeamount of a mixture of acetic and sulphuric acid. This mixture wasneutralized using solid sodium bicarbonate, a laborious task. After theisolation, the crude product had to be purified by chromatography,because the acidic hydrolysis produced a structurally closely related3-hydroxycoumarin derivative as byproduct. In conclusion: all reactionsteps of the synthetic route as outlined in EP 0650 964 show majorproblems when translated into a pilot scale production of severalkilograms of the compound.

SUMMARY OF THE INVENTION

The goal of the present invention was to overcome the abovementionedproblems, in order to be able to deliver kilogram quantities of thecompound in a safe and economically feasible way.

The problems associated with the synthesis of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one can be solved by theentirely new synthetic route according to the present invention, assummarized in the scheme below.

As a starting material a 5-halogen-2-hydroxybenzaldehyde (1) can beused, (e.g. the commercially available 5-bromo-2-hydroxybenzaldehyde),which can be selectively nitrated to yield5-halogen-2-hydroxy-3-nitrobenzaldehyde (2), the intermediate necessaryfor the Erlenmeyer condensation leading to anN-(6-halogen-8-nitro-2-oxo-2H-1-benzopyran-3-yl) amide (3) which issubsequently reduced to an N-(8-amino-2-oxo-2H-1-benzopyran-3-yl) amide(4). Rather than using the potential carcinogenN-benzyl-bis-chloroethylamine, instead a compound of the general formula

wherein L is a leaving group can be used to construct the piperazinering. This results in anN-(8-(1-piperazinyl)-2-oxo-2H-1-benzopyran-3-yl-)amide (5). Hydrolysisof this amide using an acid results in (6). It is possible to neutralizethis salt (6) to yield the free base, and to convert that into mono- ordi-acid salts. However, it has surprisingly been found that partialneutralization of the tri-hydrochloric acid salt apparently directlyresults in the monohydrochloric acid salt. Titrimetric analysis showsthat the product contains between 3.24 and 3.34 mmol HCl per gram ofproduct, the latter value being exactly the theoretical value for themono hydrochloric acid salt. ‘Karl Fisher water assay titration’ revealsthat the product of the new synthetic route contains 6.2-6.5% (mass tomass) water, for all practical purposes indicative of a monohydrate,because the theoretical value is 6% water.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention relates to a process for thepreparation of a compound of formula (7):

characterized in that a compound with formula (1):

wherein Hal is F, Cl, Br or I, is nitrated to yield5-halogen-2-hydroxy-3-nitrobenzaldehyde (2),

followed by an Erlenmeyer condensation of (2) with a glycine derivativeof formula:

wherein R represents an alkyl(C₁₋₆) or an aryl group to yield anN-(6-halogen-8-nitro-2-oxo-2H-1-benzopyran-3-yl) amide (3):

and a subsequent catalytic hydrogenation of (3) to yield anN-(8-amino-2-oxo-2H-1-benzopyran-3-yl)-amide (4),

after which the obtained compound (4) is alkylated with a compound ofthe formula

wherein L is a leaving group such as chloro, methanesulphonate orp-toluene-sulphonate, to yield anN-(8-(1-piperazinyl)-2-oxo-2H-1-benzopyran-3-yl-) amide (5),

after which the amide function of (5) is hydrolyzed using an acid,resulting in the corresponding acid salt (6), in which n is 0, 1, 2 or3.

The compound of formula (6) is further (partially) neutralized toproduce the product represented by formula (7)

in which n and m independently can be 0, 1, 2 or 3.

The halogen group used in the starting material can be selected from F,Cl, Br and I. The preferred halogen group is Br.

Examples of a leaving group L are chloro, methanesulphonate andp-toluene-sulphonate. Preferable groups chloro and methanesulphonate.The most preferred leaving group is a chloro group.

R groups are either alkyl(C₁₋₆) or aryl groups. In this description‘aryl’ means furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl,pyrazolyl, isoxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl,pyrazinyl, 1,3,5-triazynyl, phenyl, indazolyl, indolyl, indolizinyl,isoindolyl, benzi[b]furanyl, benzo[b]thiophenyl, benzimidazolyl,benzthiazolyl, purinyl, quinolynyl, isochinolyl, chinolyl, phtalazinyl,quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, naphthyl orazulenyl. Preferred R-groups are methyl and phenyl. Most preferred ismethyl.

For step 5 inorganic and organic acids can be used. Examples of acidsthat can be used are inorganic acids such as hydrochloric acid,sulphuric acid, phosphoric acid and nitric acid, or with organic acidssuch as citric acid, fumaric acid, maleic acid, tartaric acid, aceticacid, trifluoro acetic acid, benzoic acid, p-toluene sulphonic acid,methanesulphonic acid and naphthalene sulphonic acid. The preferred acidin said step is hydrochloric acid.

Step 1 can be performed in acids. The preferred solvent is acetic acid.The reaction temperature is between 0° C. and 100° C., preferably about60° C.

Step 2 can be performed in dipolar aprotic solvents. The preferredsolvent is N-methyl-2-pyrrolidone. The reaction temperature is between50° C. and 120° C., preferably about 60° C.

Step 3 can be performed in alcohols. The preferred alcohol is ethanol.The reaction temperature is between 20° C. and 100° C., preferably about60° C.

Step 4 can be performed in organic solvents. The preferred solvent ismonochlorobenzene. The reaction temperature is elevated when compared toroom temperature, preferably the reaction is carried out at reflux.

Step 5 can be performed in alcohols. The preferred solvent is ethanol.The reaction temperature is between 20° C. and 100° C., preferably about50° C.

Step 6 can be performed in alcohols. The preferred solvent is ethanol.Neutralisation is performed with a base in aqueous environment. Thepreferred base is sodium bicarbonate.

It was surprisingly found that the actual end product of one of thevariants of the new synthetic route is the mono hydrate of the monohydrochloric acid salt of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one hereafter referred to as° Compound 1′. This compound has not been described earlier and wasshown to have pharmaco-chemical properties making it suitable as a drugcandidate.

In a further aspect the invention therefore relates to novel salts andhydrates of salts of said compound, having the structure given byformula (7):

wherein ‘(acid)’ is any acid producing pharmaceutically acceptablesalts, for instance inorganic acids such as hydrochloric acid, sulphuricacid, phosphoric acid and nitric acid, or organic acids such as citricacid, fumaric acid, maleic acid, tartaric acid, acetic acid, trifluoroacetic acid, benzoic acid, p-toluene sulphonic acid, methane-sulphonicacid and naphthalene sulphonic acid, n=0, 1, 2 or 3 and m=0, 1, 2 or 3,with the proviso that when acid=HCl and n=1, m cannot be 0.

Especially preferred is the mono hydrochloric acid mono hydrate of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one.

Pharmaceutical Preparations

The compounds of the invention can be brought into forms suitable foradministration by means of usual processes using auxiliary substancessuch as liquid or solid carrier material. The pharmaceuticalcompositions of the invention may be administered enterally, orally,parenterally (intramuscularly or intravenously), rectally or locally(topically). They can be administered in the form of solutions, powders,tablets, capsules (including microcapsules), ointments (creams or gel)or suppositories. Suitable excipients for such formulations are thepharmaceutically customary liquid or solid fillers and extenders,solvents, emulsifiers, lubricants, flavorings, colorings and/or buffersubstances. Frequently used auxiliary substances which may be mentionedare magnesium carbonate, titanium dioxide, lactose, mannitol and othersugars or sugar alcohols, talc, lactoprotein, gelatin, starch, celluloseand its derivatives, animal and vegetable oils such as fish liver oil,sunflower, groundnut or sesame oil, polyethylene glycol and solventssuch as, for example, sterile water and mono- or polyhydric alcoholssuch as glycerol.

Compounds of the present invention are generally administered aspharmaceutical compositions which are important and novel embodiments ofthe invention because of the presence of the compounds, moreparticularly specific compounds disclosed herein. Types ofpharmaceutical compositions that may be used include but are not limitedto tablets, chewable tablets, capsules, solutions, parenteral solutions,suppositories, suspensions, and other types disclosed herein or apparentto a person skilled in the art from the specification and generalknowledge in the art. In embodiments of the invention, a pharmaceuticalpack or kit is provided comprising one or more containers filled withone or more of the ingredients of a pharmaceutical composition of theinvention. Associated with such container(s) can be various writtenmaterials such as instructions for use, or a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals products, which notice reflects approval by theagency of manufacture, use, or sale for human or veterinaryadministration.

Dose

The affinity of the compounds of the invention for serotonin and otherreceptors was determined as described below. From the binding affinitymeasured for a given compound, one can estimate a theoretical lowesteffective dose. At a concentration of the compound equal to twice themeasured K_(i)-value, 100% of the receptors likely will be occupied bythe compound. Converting that concentration to mg of compound per kg ofpatient yields a theoretical lowest effective dose, assuming idealbioavailability. Pharmacokinetic, pharmacodynamic, and otherconsiderations may alter the dose actually administered to a higher orlower value. The dosage expediently administered is 0.001-1000 mg/kg,preferably 0.1-100 mg/kg of patient's bodyweight.1-[2H-1-Benzopyran-2-one-8-yl]-piperazine derivatives, broad spectrum5-HT receptor binding compounds, having amongst other functionalserotonin receptor activities, potent 5-HT_(1A)-agonistic as well as5-HT_(1D)-antagonistic activity, can be used for the treatment ofaffections or diseases of the central nervous system caused bydisturbances of the serotonergic transmission, for example psychoses,aggression, anxiety disorders, autism, vertigo, disturbances ofcognition or memory, and in particular for the treatment of depression.The presence of 5-HT_(1D) antagonism is of therapeutic value. 5-HT_(1D)receptors are located presynaptically on the nerve terminal and have anegative modulatory influence on the release of 5-HT. Therefore,blockade of these receptors enhances the release of 5-HT from itsterminals. The additional presence of presynaptic 5-HT_(1D) antagonismwill result in a similar effect as observed after administration of 5-HTreuptake inhibitors. When 5-HT_(1D) antagonism is combined with5-HT_(1A) agonism the later activity is strengthened.

Surprisingly, these compounds as well as prodrugs and salts thereof,were found to be potently active in experimental animal models ofepilepsy. The compounds are devoid of sedative effects when given indosages up to 100 mg/kg p.o., and were also shown to be highly active asinducers of growth factors. The latter activity is indicative ofneuroprotective effects and improvement of brain plasticity required forneuroregeneration, and also indicative of potential therapeutic effectsin neuropathic pain (see P. Anand., “Neurotrophic factors and theirreceptors in human sensory neuropathies”, Prog. Brain Res., 146,477-492, 2004; and R. Wang et al., “Glial cell line-derived neurotrophicfactor normalizes neurochemical changes in injured dorsal root ganglionneurons and prevents the expression of experimental neuropathic pain”,Neuroscience, 121, 815-824, 2003) and diabetes induced pain (J. A.Christianson et al., “Beneficial effects of neurotrophin treatment ondiabetes-induced hypoalgesia in mice”, J. Pain, 4, 493-504, 2003).

It was also found that the compounds of the invention are active inexperimental animal models with predictive value for activity againstthe symptoms of Parkinson's disease in particular and dyskinesias ingeneral. Moreover, the compounds when given orally show a goodbioavailability, which results in high potency and long duration ofaction. The unique pharmacological profile of compounds of the inventionmakes them particularly useful in the treatment of movement disorders,including Parkinson's disease, Huntington's Chorea, progressivesupranuclear palsy, Wilson's disease, Tourette's syndrome, symptomaticand non-symptomatic epilepsy, seizures, including refractory seizuresand post-stroke seizures and other electroconvulsive disorders, variouschronic tremors, including essential tremor, tics and dystonias.

Surprisingly, these compounds as well as prodrugs and salts thereof,were also found to be potently active in experimental animal models ofpain. The pharmacological activities as realized in the compounds of theinvention and their salts represents a novel class of analgesiccompounds for the treatment of chronic pain disorders or in treatingother conditions where there is hyper-sensitization to painful signals,hyperalgesia, allodynia, enhanced pain perception, and enhanced memoryof pain.

The following examples are only intended to further illustrate theinvention, in more detail, and therefore these examples are not deemedto restrict the scope of the invention in any way.

Example I Analytical Methods Used During Syntheses

Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR spectra were recorded on a Bruker AM400 spectrometer, or a VarianVXR400S spectrometer. Chemical shifts (δ) were reported in ppm downfieldfrom TMS as internal standard. A sample of 10-50 mg was dissolved in adeuterated solvent, usually CDCl₃ or a DMSO-d6/CDCl₃ (4:1 v/v) mixture).The solvent was selected to ensure complete dissolution of the sample.The free induction decays were generally obtained at room temperatureunder the following conditions:

Digital resolution 0.2 Hz Sweep width 18 ppm Pulse width 20 degreesPulse repetition time 4.5 sec or longer if required for completerelaxation Carrier frequency 6.0 ppm Number of acquisitions 128 or moreif necessary. The C-13 satellite signals at 0.5% signal intensity shouldbe clearly visible.

NMR was used as method for determining relative contents.

Titrimetry (Chloride and Water Determinations)

For potentiometric titrations, a Metrohm model E636 (Switzerland) wasused.

Potentiometric chloride determinations were used in this syntheses todetermine chloride. The titration was performed with a combined silverelectrode and silver nitrate titrant. The method is specific forchloride because it can distinguish chloride from iodide and bromine onbasis of different electrode potentials.

Voltametric titrations for the determination according to Karl Fisherwere performed using a Metrohm 633KF (Metrohm, Switzerland) apparatusaccording to the USP method.

Example II Synthesis of 3-Amino-8-(1-Piperazinyl)-2H-1-Benzo-Pyran-2-Oneand its Monohydrochloric Acid Monohydrate

(Compound 1)

Step 1: Nitration

The first step was the nitration of 5-bromo-2-hydroxybenzaldehyde (1*)yielding 5-bromo-2-hydroxy-3-nitrobenzaldehyde (2*):

A solution of 1.0 mol of 5-bromo-2-hydroxybenzaldehyde (1*) in 3.75litres acetic acid (98%) was formed on heating the mixture to about 60°C. 1.5 mol of concentrated nitric acid (137 g=97 ml) was added slowly inapproximately 1 hour. After the completion of the addition stirring wascontinued at 65° C. for a further 10 minutes. The solution was thencooled to 45° C., and the product was precipitated by the addition of 4litres of water. After stirring for at least 3 hours the product wascollected on a filter and washed with water until the pH of the motherliquor was approximately 6. The material was dried as much as possibleby centrifugation. The crude product was dissolved in 800 ml acetoneunder refluxing and stirring. 400 ml acetone was removed bydistillation. After cooling to 20° C., the mixture was stirred for 3hours. The precipitate was collected on a filter and washed withpetroleum ether 40-65° C. The solid was dried overnight in an air streamat 40° C. Finally, the crude (2*) was recrystallized from acetone toyield an end product with a purity of 98% as shown by NMR analysis.

5-bromo-2-hydroxybenzaldehyde (1*) was identified by its characteristicchemical shift δ 9.84 ppm; 5-bromo-2-hydroxy-3-nitrobenzaldehyde (2*)had a characteristic chemical shift of δ 10.4 ppm.

The overall yield of this step was approximately 60% (crude on crude).

Step 2: Erlenmeyer Condensation

The second step was the Erlenmeyer condensation of5-bromo-2-hydroxy-3-nitrobenzaldehyde (2*) with N-acetyl-glycine toyield N-(6-bromo-8-nitro-2-oxo-2H-1-benzopyran-3-yl)acetamide (3*).

To a mixture of 1.0 mol of 5-bromo-2-hydroxy-3-nitrobenzaldehyde (2*),1.0 mol of N-acetylglycine and 1.0 mol of anhydrous sodium acetate, 800ml of N-methyl-2-pyrrolidone are added. The mixture was stirred andheated to 50° C. Then 2.2 mol of acetic anhydride was run into thereaction vessel in approximately 30 minutes. The reaction mixture washeated to 100° C. During heating the reacting mixture became homogeneousfor a while; shortly afterwards a solid was formed, making stirringtroublesome. After heating at 100° C. for 4 hours, the mixture wascooled to 80° C. and 1,100 ml of acetic acid (98%) was added. Thereafterstirring of the mixture was easy. Next, the mixture was cooled to roomtemperature, and stirred for 60 minutes. The precipitate was collectedon a filter and washed twice with 625 ml acetic acid (80%), five timeswith 900 ml water, and once with 300 ml acetone. The product was driedin an air stream at 40° C. for 24 hours, and had a purity of 98% asshown by NMR analysis.

5-bromo-2-hydroxy-3-nitrobenzaldehyde (2*) had a characteristic shift ofδ 10.4 ppm; the characteristic chemical shift ofN-(6-bromo-8-nitro-2-oxo-2H-1-benzopyran-3-yl)acetamide (3*) was δ 8.72ppm

The overall yield of this step was approximately 80% (crude on crude).

Step 3: Reduction

The third step was the catalytic hydrogenation ofN-(6-bromo-8-nitro-2-oxo-2H-1-benzopyran-3-yl)acetamide (3*) toN-(8-amino-2-oxo-2H-1-benzopyran-3-yl)-acetamide (4*).

A mixture of 1.0 mol ofN-(6-bromo-8-nitro-2-oxo-2H-1-benzopyran-3-yl)acetamide (3*), 50 g of10% palladium on carbon paste (containing 61% water), 1.0 mol ofpotassium carbonate and 15 litre of ethanol was heated to 60° C. At thistemperature the starting material was reduced with hydrogen at anoverpressure of 4 bar at 1400 rpm. After completion of the reaction (1hour), the catalyst was removed by filtration using filteraid, andwashed with 4.5 litre methyl ethyl ketone (MEK). The filtrate wasconcentrated to 2 litre, and 2.3 litre of MEK was added In order tochange the solvent from ethanol to MEK, 2 litre of the solvent mixturewas distilled off at normal pressure and 2 litre of MEK was added. Thiswas repeated 4 times. Then 5 litre of MEK and 2.6 litre of water wereadded and the mixture was stirred. The layers were separated. The upperlater was concentrated at normal pressure to approximately 3.5 litre.The residue was cooled to 25° C. During this cooling the productcrystallized. Then the mixture was cooled to −10° C. and stirred for twohours. The solid was filtered and washed three times with 800 ml hexane.The product was dried (50° C., 20 cm Hg, N₂) until constant weight.

N-(6-bromo-8-nitro-2-oxo-2H-1-benzopyran-3-yl)acetamide (3*) had acharacteristic chemical shift of δ 8.72 ppm; that ofN-(8-amino-2-oxo-2H-1-benzo-pyran-3-yl)-acetamide (4) was δ 8.55 ppm

The overall yield of this step was approximately 70% (crude on crude):

Step 4: Construction of Piperazine Ring System

Step 4 was the alkylation ofN-(8-amino-2-oxo-2H-1-benzopyran-3-yl)-acetamide (4*) withbis-chloroethylamine yieldingN-(8-(1-piperazinyl)-2-oxo-2H-1-benzopyran-3-yl-)acetamide (5*).

A mixture of 2.5 litre monochlorobenzene, 1.0 mol ofN-(8-amino-2-oxo-2H-1-benzo-pyran-3-yl)-acetamide (4*) and 1.2 molbischloroethylamine hydrochloride was heated to reflux under nitrogen.Part of the monochlorobenzene (0.5 litre) was distilled off. Thismixture was refluxed for 10 days. The reaction was followed by HPLC.After the reaction, the mixture was cooled to 20° C. and stirredovernight. The solid product was collected on a filter and washed oncewith 360 ml monochlorobenzene and 3 times with 360 ml ethanol. Theproduct was dried in vacuum at 50° C.

Half of the crude product was dissolved in 3 litre water. After additionof 18 g of Celite and 50 g of charcoal, the mixture was stirred for 1hour at room temperature. After filtration the solution was concentratedby distillation of water. In the mean time the second half of the crudeproduct was treated as described above. When the total volume of thecombined aqueous solutions was about 1.5 litre, distillation was stoppedand the mixture was cooled to room temperature. Then 125 g sodiumbicarbonate was added in portions. After stirring for 1.5 hours at 15°C. the precipitate formed was collected on a filter. After washing with360 ml water and 2 times with 270 ml ethanol, the product was dried invacuum at 50° C.

N-(8-amino-2-oxo-2H-1-benzo-pyran-3-yl)-acetamide (4*) had acharacteristic chemical shift of δ 8.55 ppm; that ofN-(8-(1-piperazinyl)-2-oxo-2H-1-benzopyran-3-yl-)acetamide (5*) was δ8.57 ppm.

The overall yield of this step was approximately 50% (crude on crude).

Step 5: Amide Hydrolysis

Step 5 was the hydrolysis of the amide function ofN-(8-(1-piperazinyl)-2-oxo-2H-1-benzopyran-3-yl-)acetamide (5*) usinghydrochloric acid. This resulted in the trihydrochloric acid salt of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one (6*).

2.9 Litre of concentrated hydrochloric acid was added at roomtemperature to a suspension of 1.0 mol ofN-(8-(1-piperazinyl)-2-oxo-2H-1-benzopyran-3-yl-)acetamide 5*) and 1.4litre of absolute ethanol in about 10 minutes. During this addition thetemperature rose to 40° C. After the addition the mixture was stirred ata temperature of 50° C. during 1.5 hours. The mixture was cooled to 20°C. and, after crystallisation had started, 1.4 litre of absolute ethanolwas added. Then the mixture was stirred for 1 hour at 20° C. and for 2hours at 0° C. The crystals were isolated by filtration and washed twicewith 0.6 litre of acetone. The isolated product was dried in vacuum (40°C., 200 mm Hg, N₂, 24 hours).

N-(8-(1-piperazinyl)-2-oxo-2H-1-benzopyran-3-yl-)acetamide (5*) had acharacteristic chemical shift of δ 8.57 ppm; the trihydrochloric acidsalt of 3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one (6*) had acharacteristic chemical shift of δ 6.77 ppm.

The overall yield of this step was approximately 85% (crude on crude).

Step 6: Partial Neutralisation

The final step, the sixth, was the partial neutralisation of thetrihydrochloric acid salt (6*) with sodium bicarbonate to produce thedesired product: COMPOUND 1, the mono hydrochloric acid mono hydrate of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one (7*)

To a suspension of 1.0 mol of the trihydrochloric acid salt (6*) in 3.5litre ethanol a solution of 2.2 mol sodium bicarbonate in 2.8 litrewater was added in about 30 minutes. The temperature was between 20° C.and 25° C. The suspension was then stirred for 3 hours. The reactionmixture was filtered and subsequently washed with 1.1 litre water, 1.1litre ethanol and 1.1 litre hexane. The isolated crude product was driedin vacuum (40° C., 200 mm Hg, N₂, 24 hours).

The dried product (1 mol) was dissolved in 9 litre methanol by heatingto reflux temperature. The solution did not become completely clear.After cooling to 20° C. the mixture was filtered. 300 ml of water and150 ml of methanol was added to the filtrate, after which about 3 litreof the solvent mixture was distilled at normal pressure. The completeprocedure was repeated with another quantity of 1 mol of the driedproduct. Then the combined fractions were concentrated to a volume ofabout 12 litres by distillation. After addition of 6 litre ethanol, 6litre of the solvent mixture was removed by distillation at normalpressure. The mixture was then cooled to 0° C. and stirred for 2 hours.The precipitate was collected on a filter and washed twice with 750 mlacetone. The product was dried under vacuum (40° C., 200 mm Hg, N₂, 24hours), and thereafter homogenized by milling and, when necessary, bymicronizing.

The overall yield of this step was approximately 85% (crude on crude).

The trihydrochloric acid salt of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one (6*) had acharacteristic chemical shift of δ 6.77 ppm; that of the endproduct,COMPOUND 1, was δ 6.7 ppm. COMPOUND 1, the mono hydrochloric acid monohydrate of 3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one, had amolecular formula C₁₃H₁₈ClN₃O₃ and a molecular mass of 299.5. The pureproduct (99.8%, NMR) was a white to yellowish powder. Its chloridecontent was 11.7% (mass to mass), as determined by titrimetry. Its watercontent, determined by Karl Fisher. water assay titration, was 6.5%(mass to mass).

Example III Formulation of Compound 1 as Used in Animal Studies

For oral (p.o.) administration: to the desired quantity (0.5-15 mg) ofCOMPOUND 1 in a glass tube, some glass beads were added and thesubstance was milled by vortexing for 2 minutes. After addition of 1 mlof a solution of 1% methylcellulose in water, the compound was suspendedby vortexing for 10 minutes. For concentrations up and above 1 mg/mlremaining particles in the suspension were further suspended by using anultrasonic bath.

Example IV Receptor Binding Profile of Compound 1

The binding data collected in the table below were either obtained byCEREP (128, rue Danton, 92500 Rueil-Malmaison, France) or at SolvayPharmaceuticals B.V., using well documented standard procedures.

RECEPTOR BINDING PROFILE OF COMPOUND 1 K_(i)(nM) receptor S¹ radioligandCompound 1 5-HT_(1A) h [³H]-8-OH-DPAT 0.25 5-HT_(1B) r[¹²⁵I]-cyanopindolol 2.0 5-HT_(1D) b [³H]-serotonin 13 5-HT_(2A) h[³H]-ketanserin 630 5-HT_(2B) h [³H]-LSD 320 5-HT_(2C) h[¹²⁵I]-DOI >1,000 5-HT₃ h [³H]-BRL 43694 250 5-HT₄ h [³H]-GR113808 >1,000 5-HT₅ h [³H]-LSD 100 5-HT₆ h [³H]-LSD >1,000 5-HT₇ h[³H]-LSD 3.2 5-HT_(reuptake) h [³H]-paroxetine >1,000 α₁-adrenergic r[³H]-prazosin >1,000 α_(1A)-adrenergic r [³H]-prazosin 630α_(1B)-adrenergic r [³H]-prazosin >1,000 α₂-adrenergic r [³H]-RX821002 >1,000 β₁-adrenergic h [³H]-CGP 12177 50 β₂-adrenergic h [³H]-CGP12177 40 β₃-adrenergic h [¹²⁵I]-iodocyanopindolol >1,000 NA_(reuptake) h[³H]-nisoxetin >1,000 Dopamine-D₁ h [³H]-SCH 23390 >1,000 Dopamine-D₂ h[³H]-spiperone >1,000 Dopamine-D₃ h [³H]-spiperone >1,000 Dopamine-D₄ h[³H]-spiperone >1,000 Dopamine-D₅ h [³H]-SCH 23390 >1,000Dopamine_(reuptake) h [³H]-GBR 12935 >1,000 Muscarine-M₁ h[³H]-pirenzepine >1,000 Muscarine-M₂ h [³H]-AFDX-384 >1,000 Muscarine-M₃h [³H]-4-DAMP >1,000 Muscarine-M₄ h [³H]-4-DAMP >1,000 Muscarine-M₅ h[³H]-4-DAMP >1,000 Histamine-H₁ h [³H]-pyrilamine >1,000 Histamine-H₂ h[¹²⁵I]-APT >1,000 Histamine-H₃ r [³H]-α-methylhistamine >10,000tryptamine r [³H]-tryptamine >10,000 melatonin c[¹²⁵I]-2-iodomelatonin >10,000 nicotine r [³H]-cytisine >10,000 μ-opiater [³H]-DAMGO >1,000 κ-opiate r [³H]-U 69593 >1,000 δ-opiate r[³H]-DPDPE >1,000 nociceptin (ORL₁) h [³H]-nociceptine >1,000 sigma r[³H]-DTG >1,000 sigma-SG₁ g [³H]-pentazocone >1,000 sigma-SG₂ r[³H]-DTG >1,000 cannabinoid-CB₁ h [³H]-WIN 55,212-2 >10,000 Ca⁺⁺-channelp [³H]-fluspirilene >10,000 Ca⁺⁺-channel r [³H]-nitrendipine >10,000Ca⁺⁺-channel r [³H]-diltiazem >10,000 Ca⁺⁺-channel r[¹²⁵I]-Ω-conotoxin >1,000 Ca⁺⁺-channel r [³H]-D-888 130 Ca⁺⁺-channel r[³H]-devapamil >10,000 Na⁺-channel r [³H]-bathrachotoxinin >10,000K⁺-channel r [¹²⁵I]-α-dendrotoxin >1,000 K⁺-channel r[¹²⁵I]-apamin >1,000 Adenosine-A₁ h [³H]-DPCPX >1,000 Adenosine-A_(2A) h[³H]-CGS 21680 >1,000 Adenosine-A₃ h [³H]-AB-MECA >1,000 Purine-P2X r[³H]-ab-MeATP >1,000 GABA_(A) r [³H]-muscimol >10,000 GABA_(B) r [³H]-PK11195 >1,000 Glycine r [³H]-strychnine >10,000Glycine_(strychn, insens.) r [³H]-MDL105519 >10,000 NMDA r [³H]-CGS19755 >10,000 angiotensin-AT1 h [¹²⁵I]-angiotensin II >1,000angiotensin-AT2 h [¹²⁵I]-CPG 42112A >1,000 benzodiazepine r[³H]-diazepam >10,000 bombesin r [¹²⁵I]-bombesin >1,000 bradykinin h[³H]-bradykinin >1,000 CCK_(A) h [³H]-devazepide >1,000 CCK_(B) h[³H]-CCK8 >1,000 CCR1 h [¹²⁵I]-MIP-1a >1,000 CGRP h [¹²⁵I]-CGRPa >1,000CRF h [¹²⁵I]-oCRF >10,000 Endothelin-ET_(A) h [¹²⁵I]-endothelin-1 >1,000Endothelin-ET_(B) h [¹²⁵I]-endothelin-1 >1,000 Galanin-GAL₁ h[¹²⁵I]-galanin >1,000 Galanin-GAL₂ h [¹²⁵I]-galanin >1,000Interleukine-6 h [¹²⁵I]-interleukine-6 >10,000 Interleukine-8 h[¹²⁵I]-interleukine-8 >1,000 LTB₄ g [³H]-LTB₄ >10,000 LTD₄ g[³H]-LTD₄ >10,000 melanocortin h [¹²⁵I]-NDP-a-MSH >1,000 Neurokinin-NK₁h [³H]-substance P >1,000 Neurokinin-NK₂ h [¹²⁵I]-neurokinin_(A) >1,000Neurokinin-NK₃ h [³H]-SR 142801 >1,000 Neuropeptide Y₁ h[¹²⁵I]-PYY >1,000 Neuropeptide Y₂ h [¹²⁵I]-PYY >1,000 Neurotensin-NT₁ h[¹²⁵I]-neurotensin >1,000 PACAP r [¹²⁵I]-PACAP 1-27 >1,000Prostaglandin-I₂ h [³H]-iloprost >1,000 Prostaglandin-H₂ h [³H]-SQ29548 >1,000 somatostatin m [¹²⁵I]-somatostatin >1,000 TRH r[³H]-TRH >10,000 Tumor necrosis f. r [¹²⁵I]-TNFα >1,000Vasopressine-V_(1A) h [³H]-vasopressine >1,000 VIP₁ h [¹²⁵I]-VIP >1,000S¹: b = bovine, c = chicken, g = guinea pig, h = human, m = mouse, p =pig; r = rat.

1. A compound of formula:

obtained by a process comprising: (a) nitrating a compound of formula(1):

wherein Hal is F, Cl, Br, or I, to yield a5-halogen-2-hydroxy-3-nitrobenzaldehyde of formula (2),

(b) subjecting (2) to an Erlenmeyer condensation with a glycine offormula

wherein R is chosen from an alkyl(C₁₋₆) and an aryl group, to yield anN-(6-halogen-8-nitro-2-oxo-2H-1-benzopyran-3-yl) amide of formula (3),

(c) subjecting (3) to a catalytic hydrogenation to yield anN-(8-amino-2-oxo-2H-1-benzopyran-3-yl) amide of formula (4),

(d) alkylating (4) with a compound of the formula

wherein L is a leaving group, to yield anN-(8-(1-piperazinyl)-2-oxo-2H-1-benzopyran-3-yl) amide of formula (5),

(e) hydrolyzing (5) with HCl, to yield the corresponding HCl salt (6),

wherein n is 2 or 3, and (f) at least partially neutralizing (6) with abase to produce the compound of formula:


2. A hydrate of a salt of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one of formula (7)

wherein the acid is chosen from organic and inorganic acids, and n* is 1or 2, and m is
 1. 3. A hydrate according to claim 2, wherein the acid isHCl.
 4. A hydrate according to claim 3, wherein n* is 1, such that thecompound is a mono hydrate of the mono hydrochloric acid salt of3-amino-8-(1-piperazinyl)-2H-1-benzopyran-2-one of formula:


5. A pharmaceutical composition comprising a pharmacologically activeamount of at least one compound chosen from the compounds of formula(7),

and salts thereof, wherein n* is 1 or 2, and m is 1, and furthercomprising at least one component chosen from pharmaceuticallyacceptable carriers and pharmaceutically acceptable auxiliarysubstances.
 6. A pharmaceutical composition according to claim 5,wherein the acid is HCl.